Embryonic Endothelial Evolution towards First Hematopoietic Stem Cells Revealed by Single-Cell Transcriptomic and Functional Analyses
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PROVIDER: PRJNA579534 | ENA |
REPOSITORIES: ENA
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Project description:Hematopoietic stem cells (HSCs) in adults are believed to be born from hemogenic endothelial cells (HECs) in mid-gestational embryos. Due to the rare and transient nature, the HSC-competent HECs have never been stringently identified and accurately captured, let alone their genuine vascular precursors. Here, we first used high-precision single-cell transcriptomics to unbiasedly examine the relevant EC populations at continuous developmental stages with intervals of 0.5 days from embryonic day (E) 9.5 to E11.0. As a consequence, we transcriptomically identified two molecularly different arterial EC populations and putative HSC-primed HECs, whose number peaked at E10.0 and sharply decreased thereafter, in the dorsal aorta of the aorta-gonad-mesonephros (AGM) region. Combining computational prediction and in vivo functional validation, we precisely captured HSC-competent HECs by the newly constructed Neurl3-EGFP reporter mouse model, and realized the enrichment further by a combination of surface markers (Procr+Kit+CD44+, PK44). Surprisingly, the endothelial-hematopoietic dual potential was rarely but reliably witnessed in the cultures of single HECs. Noteworthy, primitive vascular ECs from E8.0 experienced two-step fate choices to become HSC-primed HECs, namely an initial arterial fate choice followed by a hemogenic fate conversion. This finding resolves several previously observed contradictions. Taken together, comprehensive understanding of endothelial evolutions and molecular programs underlying HSC-primed HEC specification in vivo will facilitate future investigations directing HSC production in vitro.
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Project description:Hematopoietic stem cells (HSCs) in adults are believed to be born from hemogenic endothelial cells (HECs) in mid-gestational mouse embryos. Due to rare and transient nature, the HSC-competent ECs have never been stringently identified and accurately captured, let alone their genuine vasculature precursors. Here, we firstly used high-precision single-cell transcriptomics to unbiasedly examine relevant EC populations at continuous developmental stages and transcriptomically identified putative HSC-primed HECs. Combining computational prediction and in vivo functional validation, we precisely captured HSC-competent HECs by newly constructed Neurl3-EGFP reporter mouse model, and realized enrichment further by surface marker combination. Surprisingly, endothelial-hematopoietic bi-potential was rarely but reliably witnessed in culture of single HECs. Noteworthy, primitive vascular ECs experienced two-step fate choices to become HSC-primed HECs, resolving several previously observed contradictions. Taken together, comprehensive understanding of endothelial evolutions and molecular programs underlying HSC-primed HEC specification in vivo will facilitate future investigations directing HSC production in vitro.
2020-03-24 | GSE139389 | GEO
Project description:Whereas hundreds of endothelial cells in the aorta-gonad-mesonephros (AGM) region transdifferentiate to hematopoietic cells, only 1-2 of these cells take on hematopoietic stem cell (HSC) identity at a single time point. Gata2 is one of a few transcription factors essential for HSC generation and function. In contrast to cell surface marker-based HSC enrichment approaches, enrichment based on Gata2 expression is directly linked to the internal regulatory network critical for HSC identity. Here we use index sorting of highly enriched Gata2-expressing intra-aortic hematopoietic cluster (IAHC) cells, iterative single cell transcriptomics and functional analyses to examine the relationship between HSC identity and expression of pivotal factors. Functional HSCs are enriched in Gata2 medium-expressing IAHC cells, which can be further subdivided based on Gata2, cKit and CD27 expression, where each subpopulation is not only associated with a specific molecular profile, but also distinct functionality in both in vitro and in vivo stem/progenitor assays. Immunohistochemical analysis localizes the first putative functional HSCs in vivo to small clusters in the AGM region, thus integrating molecular, functional and spatial information about the first cells in a mammalian embryo that acquire full HSC identity.
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Project description:Iterative single cell analyses define the transcriptome of the first functional hematopoietic stem cells
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Project description:Whereas hundreds of cells in the mouse embryonic aorta transdifferentiate to hematopoietic cells, only very few establish hematopoietic stem cell (HSC) identity at a single time point. The Gata2 transcription factor is essential for HSC generation and function. In contrast to surface-marker-based cell isolation, Gata2-based enrichment provides a direct link to the internal HSC regulatory network. Here, we use iterations of index-sorting of Gata2-expressing intra-aortic hematopoietic cluster (IAHC) cells, single-cell transcriptomics, and functional analyses to connect HSC identity to specific gene expression. Gata2-expressing IAHC cells separate into 5 major transcriptomic clusters. Iterative analyses reveal refined CD31, cKit, and CD27 phenotypic parameters that associate specific molecular profiles in one cluster with distinct HSC and multipotent progenitor function. Thus, by iterations of single-cell approaches, we identify the transcriptome of the first functional HSCs as they emerge in the mouse embryo and localize them to aortic clusters containing 1-2 cells.
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Project description:Tracing the emergence of the first hematopoietic stem cells (HSCs) in human embryos, particularly the scarce and transient precursors thereof, is so far challenging, largely due to technical limitations and material rarity. Here, using single-cell RNA sequencing, we constructed the first genome-scale gene expression landscape covering the entire course of endothelial-to-HSC transition during human embryogenesis. The transcriptomically defined HSC-primed hemogenic endothelial cells (ECs) were captured at Carnegie stage 12-14 in an unbiased way, showing an unambiguous arterial EC feature with the up-regulation of RUNX1, MYB and ANGPT1. Importantly, subcategorizing CD34+CD45- ECs into CD44+ population strikingly enriched hemogenic ECs by over 10-fold. We further mapped the developmental path from arterial ECs via HSC-primed hemogenic ECs to hematopoietic stem progenitor cells, and revealed a distinct expression pattern of genes that were transiently over-represented upon the hemogenic fate choice of arterial ECs, including EMCN, PROCR and RUNX1T1. We also uncovered another temporally and molecularly distinct intra-embryonic hemogenic EC population, which was detected mainly at earlier CS 10 and lacked the arterial feature. Finally, we revealed the cellular components of the putative aortic niche and potential cellular interactions acting on the HSC-primed hemogenic ECs. The cellular and molecular programs and interactions that underlie the generation of the first HSCs from hemogenic ECs in human embryos, together with distinguishing HSC-primed hemogenic ECs from others, will shed light on the strategies for the production of clinically useful HSCs from pluripotent stem cells.
2019-09-09 | GSE135202 | GEO